PROJECT SUMMARY/ABSTRACT The intestinal lymphatic system serves physiological functions, including uptake of lipids, vitamins, immune surveillance, and fluid homeostasis. It is made up of distinct lymphatic endothelial cell populations that require regulatory pathways to maintain lymphatic vessel fate, microenvironment and functionality. We have demonstrated that Notch functions in cell fate determination of lymphatic endothelial cells and to maintain intestinal lymphatic homeostasis. Notch signaling is active throughout the intestinal lymphatic system, including the ductal, valve, and lacteal lymphatic endothelium. Disrupting Notch signaling or Mmp14 leads to shared lymphangiogenic and mesenteric lymphatic valve defects. Notch and MMP14 are linked to diverse intestinal and cardiovascular disorders and our novel studies will shed light on their role in intestinal diseases that have lymphatic defects as an underlying factor. We hypothesize that Notch regulates the extracellular matrix of the intestinal and mesenteric lymphatic vasculature via induction of MMPs and regulation of matrix proteins in a context-dependent manner. To address this hypothesis, we will determine lymphatic endothelial Notch function in postnatal intestinal and mesenteric lymphatics. We will use transgenic mice to transcriptional profile Notch responses in intestinal and mesenteric lymphatic endothelial cells and in a tether-ligand assay. The mechanism by which Notch regulates Mmp14 expression and its effects on lymphatic endothelial cell behavior will be determined. Conditional mice will be used to alter lymphatic endothelial Notch signaling with and without lymphatic endothelial Mmp14 deletion to define Notch functions in maturation and homeostasis of mesenteric lymphatic collecting ducts. As Notch is active in lacteal endothelial cells and Notch mutants display lacteal defects, we will use conditional mouse models to study the role of Notch in the lacteals during development and homeostasis. We will determine if abnormal matrix accumulation in the villi in Antxr1-/-;Antxr2-/- mice leads to lymphatic dysfunction and changes lymphatic endothelial Notch signaling. Our proposed research aims to uncover mechanisms by which Notch modulates extracellular matrix proteins and regulatory pathways in lymphatic endothelial cells to understand digest tract lymphatic development, homeostasis and function.